De novo germ line mutations are a major cause of infant mortality, developmental disability, and psychological disorders. In order to design therapies to prevent or treat diseases caused by de novo mutation, an understanding of the molecular underpinnings of mutagenic processes is required. Recent breakthroughs in single cell amplification and whole genome deep sequencing have brought this goal within reach. However, one of the greatest challenges is to distinguish bona fide de novo mutations from experimental noise and to do so in an unbiased way. There are two key questions that need to be addressed: What are the principal mechanisms of mutagenesis and their relative contribution to loss of genome integrity? And what features of genomic sequence or architecture render regions prone to mutation? I will develop an innovative program that can directly determine and verify de novo mutations genome-wide using unique features of germ line developmental biology and meiotic recombination in male mice. This proposal will establish a technique to sequence all four haploid cells that derived from the same meiosis, a tetrad. Further, we will isolate independent tetrads derived from the same spermatogonial stem cell lineage. Using this innovative approach, I will investigate the consequences to germ line genome integrity due to 1) loss of mismatch repair, 2) ectopic expression of activation-induced cytidine deaminase (AID), and 3) replication stress induced by the therapeutic, hydroxyurea. Taken together, the successful execution of the proposed research will provide a comprehensive understanding of what, when, where, and how mutations arise and determine how genomic sequence, chromosome architecture, and DNA repair pathways influence germ line genome integrity. Further, the establishment of this powerful technique will provide an invaluable tool for the research and medical community to delineate complex mutagenic effects derived from genetic predisposition or environmental and therapeutic exposure.